Mitochondria to plasma membrane redox signaling is essential for fatty acid β-oxidation-driven insulin secretion

We asked whether acute redox signaling from mitochondria exists concomitantly to fatty acid- (FA-) stimulated insulin secretion (FASIS) at low glucose by pancreatic β-cells. We show that FA β-oxidation produces superoxide/H2O2, providing: i) mitochondria-to-plasma-membrane redox signaling, closing KATP-channels synergically with elevated ATP (substituting NADPH-oxidase-4-mediated H2O2-signaling upon glucose-stimulated insulin secretion); ii) activation of redox-sensitive phospholipase iPLA2γ/PNPLA8, cleaving mitochondrial FAs, enabling metabotropic GPR40 receptors to amplify insulin secretion (IS). At fasting glucose, palmitic acid stimulated IS in wt mice; palmitic, stearic, lauric, oleic, linoleic, and hexanoic acids also in perifused pancreatic islets (PIs), with suppressed 1st phases in iPLA2γ/PNPLA8-knockout mice/PIs. Extracellular/cytosolic H2O2-monitoring indicated knockout-independent redox signals, blocked by mitochondrial antioxidant SkQ1, etomoxir, CPT1 silencing, and catalase overexpression, all inhibiting FASIS, keeping ATP-sensitive K+-channels open, and diminishing cytosolic [Ca2+]-oscillations. FASIS in mice was a postprandially delayed physiological event. Redox signals of FA β-oxidation are thus documented, reaching the plasma membrane, essentially co-stimulating IS.

exon-3 to eliminate the XbaI restriction site (Fig. S1).DNA from tails of young mice was analyzed by PCR restriction-fragment-length polymorphism.Purified PCR products were digested with XbaI (ThermoFisher, Waltham, MA, USA).To select appropriate mice for breeding, the PCR products were subcloned into the pGEM®-T Easy Vector System (Promega, Madison, WI, USA).Sequencing only selected those mice bearing a 13 base-pair long deletion in exon-3 for further breeding and was performed using M13 reverse and forward sequencing primers.This deletion produces a premature stop codon in the fourth exon, making it unable to express Pnpla8.The wild-type (wt) mice used were those backcrossed >10 generations into the PNPLA8-knockout mice background.Part II Meaning of experimental data for islet perifusion for FASIS and GSIS in iPLA2KO murine PIs Islet perifusion data are expressed in the same way as the direct output of the experiment.Formally and for simplicity, the y-axis units of the perifusion time courses are designated as "ng/gDNA", referring to a yield of insulin, quantified as ng of insulin per number of islet cells, which contain 1 g of DNA.However, since we actually quantified the amount of insulin secreted during the preceding 2 min for each time point, these data, representing an instant yield, can be recalculated into insulin release rates.The correct dimension of the y-axis units in the experimental plot displaying the instant yield is ng of insulin accumulated within the 2-min interval of perifusion sampling, normalized to the number of islet cells, which contains 1 g DNA (i.e. units of ng per 2 min•gDNA).Moreover, time intervals were set to 5 min after 25 min, hence the correct dimension of the y-axis units is ng of insulin accumulated within the 5-min interval of perifusion sampling, normalized to the number of islet cells, which contains 1 g DNA (i.e. units of ng/5 min•gDNA).When integrated over a chosen time-period (equal to the period of the 1 st phase or arbitrarily selected for the 2 nd phase), the resulting AUCs are expressed in units, which are truly ng/gDNA.
Fig. S2 shows how the plots for FASIS (Fig. S2A, recalculated from Fig. 1A) are re-plotted as insulin secretion rates at the given moment.Next, data for glucose-stimulated insulin secretion (GSIS) are present for murine PIs from wt and iPLA2KO mice (Fig. S2B, recalculated from original data displayed in Fig. S2C).These data demonstrate that the peak rate for wt PIs is similar for FASIS and GSIS.Fig. S2D then shows the calculated AUCs.
Also, we compare AUCs for selected FAs (Fig. S2E,F) at the doses theoretically giving similar free FA concentrations.To calculate them (Table S2), we used the following formula:   Therefore, we present here a typical example of records (Fig. S4A-C) and derived values of pentaplicates of respiratory rates for a single isolation of wt islets (Fig. S4D-F) and iPLA2 KO islets (Fig. S4G-I).They were selected from altogether N=5 isolations of islets and up to n=15 single rates evaluations for each condition, which were used to calculate phosphorylating to non-phosphorylating respiration ratios of Fig.  Insensitivity of GSIS to CPT1 silencing: Next, we validated the results of FASIS inhibition after CPT1 silencing by checking any effects of this silencing to GSIS.As Fig. S5B demonstrates, there was no effect to GSIS with added 25 mM glucose after twice washing in KRH without glucose for 15 min.

Part VI Monitoring of cytosolic redox signal upon GSIS vs. FASIS and induction of insulin secretion with pro-oxidants
Comparison of H2O2 release for different fatty acids and doses: The precise quantification of H2O2 release to the PI exterior was performed using the two main modes.In the first mode, we excluded any contact and possible interaction of Amplex UltraRed and horseradish peroxidase (HRP) with the islet surface to prevent possible artifacts.Thus, only after perifusate collection, were Amplex UltraRed and HRP added.The other advantage of this mode was perfect time matching and identity with the perifusion experiments surveying insulin secretion.The comparison of selected FAs at total FA concentrations theoretically yielding ~2.5 nM free FAs (according to Table S2) are shown in Fig. 5E, including empirically adjusted concentrations for lauric, decanoic and hexanoic acids.
The second method used a closed cuvette space, when allowing the contact of Amplex UltraRed and HRP with PIs, continually recording the accumulated Amplex UltraRed fluorescence (Fig. 5F-J).For this method, differential saturated rates of H2O2 release to PI exterior are compared in Fig. S6 for given FAs and their doses.Validation of Amplex UltraRed method for quantification of H2O2 release: The precise quantification of H2O2 release to the extracellular space is difficult in a heterogeneous and compact cell cluster like the pancreatic islet.A perifusion method might ensure that a majority of the released H2O2 could remain in the perifusate and, if reacted with Amplex UltraRed and HRP in the already collected samples, this neglects any interfering effects of e.g.islets, membranes, and column material.Each 2 min collection of perifusate fluorescence makes it possible to estimate purely the rate of H2O2 release to the extracellular space for the preceding 2 min (5 min after 25 min).Differences between samples with and without PA allowed us to determine differential () rates, corresponding to a net redox signal.
Also, we have previously demonstrated that NADPH oxidase-4 (NOX4) is the main redox signal source upon GSIS [36].Now, when introducing the method of H2O2 monitoring in the PI cell exterior with Amplex-UltraRed plus HRP, we can validate this method relative to the previously reported H2O2 elevation in the cytosol [36].Indeed, upon the addition of 25 mM glucose to PIs incubated in the cuvette (Fig. 5J), the H2O2 release to the islet cell exterior was significantly elevated and was almost entirely dependent on the presence of NOX4 (Fig. 5J).Note that the C57BL/6J progeny was used for the construction of NOX4 KO null mice, unlike a current progeny based on C57BL/6N mice and employed for the construction of iPLA2KO mice.

Indirect validation of Amplex UltraRed method for quantification of H2O2 release:
We also used data of Fig. 5L together with the quantified respiration of 63] to calculate the % yield of H2O2 vs.
consumed oxygen, which was 0.47% ±0.2% of oxygen without PA and increased to 3% ±0.4% with PA (Fig. S7A).This represents an expected reasonable value, showing that at least the order of magnitude is valid for estimated H2O2 release rates.For islets we obtained the same order of magnitude when we took data such as Fig. S4 and Fig. 5. Being unable to assay respiration and H2O2 simultaneously, we omit such data.
Monitoring of the cytosolic redox signal: Upon FASIS, INS-1E cells also exhibited cytosolic H2O2 release, monitored with DCF (Figure S7B,C), or with HyPer-7 (Fig. S7E).Since the DCF probe is a relatively nonspecific ROS indicator, sensing ROS downstream of H2O2, we omitted its calibration.Nevertheless, 10 nM SkQ1 inhibited its increasing fluorescence after PA addition (with 15 μM BSA), indicating the absence of the redox signal (Fig. S7B,C).The redox signal was also absent with only 1 μM Agonist II without FA (Fig. S7B-D).Also, INS-1E cells transfected with the cytosolic HyPer-7 fluorescence probe yielded minor, but statistically significant, increases in fluorescence intensity ratios at 488 nm vs. 405 nm upon a standard cell FASIS test (15 μM palmitic acid, with 15 μM BSA) (Fig. S7E).
Rescue of cytosolic redox signal: Previously, we also demonstrated that the lack of NOX4, disabling GSIS, can be rescued with the externally added H2O2 to PIs of NOX4 KO mice (cf. Figure 4M,N in [36]).Also, insulin release in INS-1E cells induced by tert-butylhydroperoxide (tBHP) reached ~70% of GSIS (cf.Fig. 7B in [37]).In iPLA2-silenced INS-1E cells, the tBHP-induced insulin release was even higher due to the absence of antioxidant uncoupling otherwise provided by a synergy of UCP2 plus iPLA2 [37].We conducted similar experiments in wt and iPLA2KO-PIs using H2O2 (Fig. 5N-P).We also present here the induced response of PIs by tBHP, manifested as a relatively low 1 st phase of insulin release in wt-PIs but not iPLA2KO-PIs; followed by a delayed "rescue" at 30 min in both wt as well as in iPLA2KO-PIs (Fig. S7F).A negligible inhibition by GW1100 was observed for such a "rescue" by H2O2 (Fig. S7G), while AUCs for both phases taken together are listed in Fig. S7H (including more data as well as the data of Fig. 5N-P; N=3-6).when "no addition rates" were subtracted).Where indicated, 10 nM SKQ1 together with PA (cyan) or 1 μM Agonist II without PA were used.Note that the sole 1 μM Agonist II had no effect even when using exterior H2O2 release monitoring with Amplex UltraRed (D).(E) HyPer-7 fluorescence intensity ratios at 488 nm vs. 405 nm in standard cell FASIS test (15 μM palmitic acid, i.e. 1.4 nM free with 15 μM BSA) prior to (0 min) and 10 min after palmitic acid addition.Data collection from 9 cell passages (averages for each biological replicate are indicated) containing estimations of 66 cells.F-H) Insulin secretion stimulated by tert-butylhydroperoxide (tBHP) (F) and H2O2 (G,H) at low glucose (5.5 mM) -wt-PIs (black, violet) and iPLA2KO PIs (green, green/violet semifilled symbols) were perifused in the presence of 100 μM tBHP (F) or 100 μM H2O2 (G,H).Where indicated, 1 M GW1100 (black/cyan or dark green/white semi-filled symbols) was present (G); or 2.5 M etomoxir (red, dark red) was tested (H).Part VIII GSIS and absence of insulin resistance in iPLA2KO mice GSIS was not significantly different in wt and iPLA2KO mice (Fig. S9A-D), while the knock-outs did not exhibit any peripheral insulin resistance (Fig. S9E,F).GSIS was tested in mice either after i.p. glucose administration, as described previously [36] (Fig. S9A,B), or after oral glucose administration (Fig. S9C,D).
Statistically, with a low significance, the GSIS 2 nd phase in iPLA2KO mice appeared to be somewhat higher (Fig. S9C), which may reflect a mild impaired glucose tolerance.Figure S9E Insulin resistance was assayed as 14 C-glucose uptake into lipids of epidydimal adipose tissue (for method, see Ref. 36 ), sensitive to insulin (differences between with and without insulin were assessed; n = 9).
For i.p. administration, the 1 st GSIS phase peaked at around 10 min in wt (backcrossed) mice (Fig. S9A) and was slightly higher (P < 0.05) in iPLA2KO mice (AUC 1st 148%-162% of wt AUC 1st ).The 2 nd GSIS phase was even higher in some individual iPLA2KO mice (AUC 2nd 257%-284% of wt AUC 2nd ).With glucose administered orally, i.e. providing a test that also includes responses to incretins, the data for an ensemble of iPLA2KO mice yielded an overall earlier initial insulin rise with a wider peak relative to wt mice, in which a sharper peak of the GSIS 1 st -phase was observed.The 2 nd phases clearly exhibited glycemia (Fig. S9C,D), decaying similarly in wt and iPLA2KO mice.
These data already suggest that no significant glucose intolerance was developed in iPLA2KO mice, despite slightly but statistically insignificantly higher values than wt mice (Fig. S9B).When assayed as the insulin-induced 14 C-glucose uptake into epidydimal white adipose tissue of iPLA2KO vs. wt mice, no significantly developed insulin resistance was found (Fig. S9E).

Part IX Details of in vivo FASIS in wt vs. iPLA2KO mice
First, we will discuss whether three apparent phases of insulin secretion after oral administration of Intralipid to mice are only delayed in iPLA2KO mice.We have calculated AUCs from the data of Fig. 9A, while taking averages ± SDs and recognizing three phases for wt mice (0-20 min; 20-240 min; and >240 min) and four phases for iPLA2KO mice (0-15 min; 15-60 min; 60-240 min; and >240 min) (Fig. S10A).This sectioning complies with the assumption that FASIS is only delayed in iPLA2KO mice, specifically the 1 st wt phase is shifted to the 15-60 min of iPLA2KO mice.
Second, the ratio of molecular weights for murine c-peptide (3121.41) vs. insulin (5803.6) is 0.537840306.Calculating this ratio for their estimated values for each individual mouse provides more precise insight into physiological postprandial events.Whenever this ratio exceeds the true stoichiometry of 0.5378, insulin was internalized/degraded faster from the blood relative to c-peptide.This internalization is supposed to be maximum when an insulin secretion initiates again.Hence, the coincidence of c-peptide to insulin peaks with the insulin peaks supports the new starting insulin secretion at the given moments.
Third, in order to determine whether insulin secretion stimulated upon the initial administration of either palmitic acid (Fig. 8A,C), Intralipid (Fig. 8B,D,F; Fig. 9A-E), or Agonist II (Fig. 8I) may contain a component given by the endogenously elevated glucose exceeding the initial fasting levels in mice, we recalculated experimental data of blood insulin and glycemia to divide the secreted insulin amounts by the glycemia in the given period (units in ng per mmol).When the glucose (glycemia) does not contribute to insulin secretion, the resulting time course is like that for insulin alone.This was specifically the case for longer periods from the initial oral lipid administration (Fig. S10A).With Agonist II i.p. administration, data reflect its modest ability to stimulate insulin secretion at low glucose (Fig. S10B,C).With GSIS, normalized peaks of insulin secretion are only transformed into small elevations of insulin/glycemia values (Fig. S10D,E).

Figure S1 (
Figure S1 (A) Construction of PNPLA8 knock-out mice by targeting the PNPLA8 gene, exon 3, using TALEN (FokI nuclease).Its cleavage deletes 13 base pairs, including the XbaI restriction site and creates a premature stop codon in exon 4. (B) Verification by PCR restriction-fragment-length polymorphism of the genomic DNA purified from mouse tails; +/+ wild type, +/-heterozygote, -/-knock-out.Primers PNPLA8 forward were AAGAGTCCGCCCGAAGAACAG and PNPLA8 reverse AGGCTTTGCATTCCCCACTTT.XbaI restriction provides a wt band of ~400, whereas the KO band is ~500.

Figure S2
Figure S2 Perifusion data recalculated as insulin secretion rate at the given moment, comparison of various FAs -Raw experimental data of Fig. 1A for FASIS and panel C for GSIS were recalculated to obtain the instant rate of insulin secretion (A,B), calculated as the average rate over the preceding 2 min (preceding 5 min after 25 min of the run).PIs of backcrossed wt mice (black, or semi-filled symbols) and iPLA2KO mice (green) were perifused as described in the Fig. 1A legend.When indicated, 1 M GW1100 (black/cyan or dark green/white semi-filled symbols) was present.D) Comparison of AUCs for FASIS and GSIS.(E,F) Total insulin amounts secreted at the 1 st phase (E) and 2 nd phase (F) upon PI perifusion with FAs giving equal H2O2 response in 30 μM BSA, i.e.50 μM PA, wBr-PA, linoleic; 65 μM stearic; 70 μM oleic; 90 μM lauric; and 180 μM hexanoic acid.

Figure
Figure S4 Respiration rates corrected for nonmitochondrial respiration -compared wt vs. iPLA2KO pancreatic islets.A-C) Representative records of Agilent Seahorse XF 24 analyzer for pentaplicates (N=5) of a typical single islet isolation.D-I) Derived respiratory rates, corrected for non-mitochondrial respiration.Panels A-C) compare respiration rates in the absence of palmitic acid to the rates A) in the presence of 50 M palmitic acid with 30 M BSA; B) with 10 nM SkQ1 in addition to 50 M PA (+30 M BSA) and C) with 2.5 M etomoxir in addition to 50 M PA (+30 M BSA).Panels D-I) sort the derived respiration rates according to the state, either phosphorylating respiration rates (V3), non-phosphorylating respiration rates, set with oligomycin (V4) and roughly derived maximum rates in the presence of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; VFCCP).
1N.Oligomycin was used to set the non-phosphorylating respiration.The minimum OXPHOS values at 5.5 mM glucose in the absence or presence of etomoxir in iPLA2KO PIs (Fig.1N) then reflect the absence of endogenous FAs cleaved from mitochondria, as expected in the absence of phospholipase.Probably in wt PIs these endogenous FAs cleaved by phospholipase with non-zero activity (but not yet activated to maximum) contribute to the observed higher OXPHOS intensity.Part V Carnitine palmitoyl transferase 1 silencingVerification: The Silencer Select siRNA for carnitine palmitoyl transferase 1 (ThermoFisher, ID: s130674 Cpt1a) was verified by PCR for its efficiency 48 hours after transfection.The relative expression ratio (decrease) was calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown "silenced" sample vs. control parental INS-1E cells and cells transfected with siRNA bearing the scrambled sequence, using formula:Decrease = (Etarget) CPtarget (control -sample) / (Eref)CPref (control -sample) {eq.2}, from the reference [73], Pfaffl MW.Nucleic Acids Res.2001;29(9):e45.Etarget is the PCR efficiency of target gene transcript, while Eref is the PCR efficiency of the used reference gene.CPtarget is a crossing point deviation between control and sample for the target gene transcript, while CPref stands analogically for reference gene.We performed PCR for a CPT1 amplicon with a forward primer CTGTCAACCTCGGACCCAAA and a reverse primer CAGCATCTCCATGGCGTAGT. From the data obtained by PCR and using the eq.{2}, we have obtained the relative decrease of CPT1 amplicon down to 20% for treatment of INS-1E with s130674Cpt1a (Fig.S5A).The control housekeeping gen was rat YWHAZ, amplified with a forward primer AGCCCGTAGGTCATCTTGGA and a reverse primer with sequence of TGCGAAGCATTGGGGATCAA. Realtime qPCR was performed on a Biorad CFX Connect, using protocol with 50 cycles, temperatures 95 o C, 59 o C, 72 o C, timing 7 s, 10 s, 15 s, respectively.RNA isolation was done using the RNA Qiagen RNeasy mini kit..

Figure S5 .
Figure S5.Verification of CPT1 silencing and insensitivity of GSIS to CPT1 deficiency -A) The relative content of CPT1 amplicon, calculated using eq.{2} for parental INS-1E cells (black), cells transfected with scrambled siRNA (gray) and cells transfected with s130674 Cpt1a.B) GSIS: 3 mM (gray, dark red) or 25 mM glucose (black, red) was added to INS-1E cells after twice preincubation in KRH with no glucose and accumulated insulin was assayed.The calculated differential rate of insulin secretion (slope of samples with 3 mM glucose subtracted) after CPT1 silencing accounted for 112% of the rate in controls, transfected with scrambled siRNA.

Figure
Figure S6External H2O2 release from pancreatic islets monitored with Amplex UltraRed & HRP in a fluorometer cuvette -compared are rates of H2O2 release with subtracted rates with no FA added for selected FAs and their indicated total concentrations.The rates were taken when saturated during the time interval of 6 to 10 min and were normalized to those measured with 50 M palmitic acid (PA)
of Ca 2+ oscillations to glucose in wt vs. iPLA2KO murine PIsWe compared oscillations in cytosolic Ca 2+ responding to palmitic acid (Fig.7) to those at increasing glucose between 3 mM and 20 mM, monitored in cells with an expressed slow variant of the GCaMP6 fluorescence probe by confocal microscopy (Fig.S8).Fluorescence emission was collected from ROI within each single responding cell.Observed cytosolic Ca 2+ -oscillations [Ca 2+ ]c(t) at a given increasing glucose differed in peak intensity histograms.At 3 mM glucose, over 60% of peaks were of the smallest intensity, falling into the lowest decile (i.e.10-percentile) of the whole intensity range, with other peaks falling in the second and third smallest bin.At 5 mM glucose, the [Ca 2+ ]c(t) spikes with ~ 20% of maximum intensity were most frequent and at 9 mM glucose those with ~ 50% of maximum intensity were most frequent, while in both cases even higher intensities were abundant.

Figure
Figure S8A Histograms of intensities of Ca 2+ -oscillations, binned in deciles for INS-1E cells, induced with increasing glucose concentrations, indicated in each panel.Around 1000 Ca 2+ -oscillation peaks were taken.

Figure S8B
Figure S8B Theoretical histograms of intensities of Ca 2+ -oscillations for nimodipine and exp.results of catalase overexpression, binned in deciles for INS-1E cells.Here the first decile was divided into noise (below 5% of the maximum signal) and the remaining first decile.Around 20 min of single-cell records were analyzed with nimodipine plus PA, and 10 min with PA and catalase overexpression.

Figure S9 .
Figure S9.GSIS in wt vs. KO mice -intraperitoneal (i.p.) administration of glucose (1 mg/g body weight; ~111 mol glucose per mice) (A,C) or oral administration of glucose (1 mg/g body weight) (B,D) was performed with wt (black) and iPLA2/PNPLA8 KO mice (green).At 2-3 time points (i.e.estimates of insulin and glycemia, organized so to cover 60 min), blood was sampled from the eye plexus blood vessel for each mouse and resulting insulin time courses (A,B) and glycemia time courses (C,D) were constructed.Average values are connected by thick lines, while all measured time points of several experiments are shown.The time dependencies were constructed from numerous groups of mice of different littermates, both males and females (9 and 32 mice in each group for A,C and B,D, respectively) covering all the tested time points, while the different littermates are indicated with different symbols.This setup is the only way to perform fast sampling of insulin release using the most sensitive insulin Elisa kit (Mercodia, Uppsala, Sweden).Student's T-test calculated for each time point individually yielded non-significant differences except for pairs marked with: ** P < 0.05; * P < 0.1.In panel D, all data up to 20 min were statistically significant (P < 0.05) relative to saline-only data.FigureS9EInsulin resistance was assayed as 14 C-glucose uptake into lipids of epidydimal adipose tissue (for method, see Ref.36 ), sensitive to insulin (differences between with and without insulin were assessed; n = 9).

Figure S10 .
Figure S10.Parameters of insulin secretion stimulated in mice with oral Intralipid and i.p. administered Agonist II -(A) AUCs for insulin time courses up to 360 min in three phases after oral administration of Intralipid (data of Fig. 9A); (B, C) GSIS (i.p. glucose) for comparison -parameters derived as C-peptide to insulin ratios (B) and insulin to glycemia ratios (C) from the data of Fig. 9D-F.D -G) Parameters of insulin secretion in mice after i.p. administration of Agonist II (data of Fig. 8I).